I. Field of the Invention
The present invention provides a process for protein purification, specifically a process for purifying alpha-1 proteinase inhibitor (or alpha-1 antitrypsin or "API") from blood plasma or blood plasma fractions. The present invention further provides formulations of 100% active API.
II. Technology Background
Alpha-1 proteinase inhibitor is a glycoprotein having molecular weight of 53,000. API has a role in controlling tissue destruction by endogenous serine proteinases, and is the most pronounced serine protease inhibitor in blood plasma. In particular, API inhibits various elastases including neutrophil elastase. See Biochem. Biophys. Res. Comm., Vol. 72, No. 1, pages 33-39, (1976); ibid., Vol. 88, No. 2, pages 346-350, (1979).
Elastase is a proteinase which breaks down tissues, and can be particularly problematic when its activity is unregulated in lung tissue. This protease functions by breaking down foreign proteins. However, when API is not present in sufficient quantities to regulate elastase activity, the elastase breaks down lung tissue. In time, this imbalance results in chronic lung tissue damage and emphysema. In fact, a genetic deficiency of API has been shown to be associated with premature development of pulmonary emphysema. API replenishment has been successfully used for treatment of this form of emphysema.
A deficiency of API may also contribute to the aggravation of other diseases such as cystic fibrosis and arthritis, where leukocytes move in to the lungs or joints to fight infection. With a deficiency in API, uninhibited lymphocyte elastase may result in destruction of the surrounding tissue. Thus, API could conceivably be used to treat diseases where an imbalance between inhibitor and protease(s), especially neutrophil elastase, is causing progression of a disease state. Antiviral activity has also been attributed to API.
Purified API has been approved by the FDA for chronic replacement therapy in individuals with congenital deficiency for the treatment of panacinar emphysema. The existing API market is under-supplied; while research suggests additional therapeutic uses for API. For example, studies suggest API might be useful against tissue destruction by uncontrolled neutrophil elastase activity. Such research is hindered by lack of supply. More efficient means of isolation, or alternative sources, of API are required.
Several groups have reported production of recombinant API. See, e.g., G. Wright et al., "High Level Expression of Active Human Alpha-1-Antitrypsin in the Milk of Transgenic Sheep", Biotechnology, Vol. 9, pp. 830-834 (1991); and A. L. Archibald et al., "High-level Expression of Biologically Active Human Alpha-1-Antitrypsin in the Milk of Transgenic Mice", Proc. Nat'l. Acad. Sci. USA., Vol.87, pp. 5178-5182 (1990). However, human plasma is the only approved source of therapeutic API. Anticipated API demand is shown in Table 1.
TABLE 1 ______________________________________ Anticipated demand for API. Treatment (& type of # Yearly Product administration) Patients Dose (g) Needs (kg) ______________________________________ Congenital 1,700 218 370 Deficiency (Intravenous) Cystic Fibrosis - 30,000 75 2,250 U.S. (Aerosol) COPD, ARDS, &gt;5,000,000 variable large Emphysema (not PiZZ) (Aerosol).sup.1 Psoriasis (Topical) 235,000 12 2,830 ______________________________________ .sup.1 COPD = Chronic Obstructive Pulmonary Disease ARDS = Adult Respiratory Disease Syndrome PiZZ = hereditary form of API deficiency resulting from a point mutation in the gene for API
Combined human plasma sources produce approximately 7,000,000 L of human plasma per year. Every one million liters of plasma produces about 20,000 kg of Cohn Fraction IV-1 paste. And every kg of Cohn Fraction IV-1 paste contains between 15 and 20 g of API. Thus, even if API could be isolated at 100% efficiency and activity from all available human sources, there would still not be enough to meet demand.
As it is, existing isolation and purification processes are inadequate. Trace impurities resulting from inefficient purification processes can stimulate an immune response in patients. Furthermore, purification processes that fail to separate active and inactive API can lead to a product with unpredictable efficacy and a specific activity which varies between separate lots. PROLASTIN.TM. (Miles, Inc.) is the only plasma-derived, FDA-approved product presently on the market. However, this product is not completely pure, and typically contains about 12% albumin and 2.5% IgA, and is only 60% active.
A number of methods have been employed to isolate API from the blood plasma. A majority of these methods are directed to laboratory scale isolation while others pertain to production on a commercial level. Several methods of isolation are summarized in U.S. Pat. Nos. 4,379,087 and 5,610,285, which are incorporated by reference. Many early methods employed ammonium sulfate precipitation from human plasma and dialysis, followed by a subsequent chromatographic step on DEAE-cellulose. However, dialysis is not easily applicable to large scale purification, and is a lengthy, time consuming process likely to compromise activity of the isolated protein.
A large scale purification of API from human plasma was disclosed by Kress et al., Preparative Biochemistry, Vol. 3, No. 6, pages 541-552 (1973). The precipitate from the 80% ammonium sulfate treatment of human plasma was dialyzed and chromatographed on DEAE-cellulose. The concentrate obtained was again dialyzed and gel filtered on SEPHADEX.RTM. G-100. The API-containing fractions were chromatographed twice on DE-52 cellulose to give API.
Glaser et al., Preparative Biochemistry, Vol. 5, No. 4, pages 333-348 (1975) isolated API from Cohn Fraction IV-1. In this method, dissolved IV-1 was chromatographed on DEAE-cellulose, QAE-SEPHADEX.RTM., concanavalin A-SEPHAROSE.RTM., and G-150 SEPHADEX.RTM. to give API. However, Glaser et al. achieved only a 30% overall yield.
Podiarene et al., Vopr. Med. Khim. 35:96-99 (1989) reported a single step procedure for isolation of API from human plasma using affinity chromatography with monoclonal antibodies. API activity was increased 61.1 fold with a yield of only 20%.
Burnouf et al., Vox. Sang. 52: 291-297 (1987) starting with Cohn Fractions II+III used DEAE chromatography and size exclusion chromatography to produce an API which was 80-90% pure (by SDS-PAGE) with a 36-fold increase in purity. Recovery was 65-70% from the supernatant A.
Hein et al., Eur. Respir. J. 9: 16s-20s (1990) presented a process that employs Cohn Fraction IV-1 as the starting material and utilized fractional precipitation with polyethylene glycol followed by anion exchange chromatography on DEAE SEPHAROSE.RTM.. The final product has a purity of about 60% with 45% yield.
Dubin et al., Prep. Biochem. 20: 63-70 (1990) used a two step chromatographic purification whereby alpha-PI, C.sub.1 -inhibitor, alpha-1 antichymotrypsin, and inter alpha-1 trypsin inhibitor were first eluted from Blue SEPHAROSE.RTM. and then API was purified by gel filtration. Purity and yield data were not given.
Jordan et al., U.S. Pat. No. 4,749,783 (1988) described a method where biologically inactive proteins in a preparation were removed by affinity chromatography after a viral inactivation step. The basis of the separation between the native and denatured forms of the protein was the biological activity of the native protein towards the affinity resin and not physical differences between the native and denatured proteins.
An integrated plasma fractionation system based on polyethylene glycol (PEG) was disclosed by Hao et al., Proceedings of the International Workshop on Technology for Protein Separation and Improvement of Blood Plasma Fractionation, Sept. 7-9, 1977, Reston, Va. In the published method Cohn cryoprecipitate was mixed with increasing concentrations of PEG in order to obtain four different PEG fractions. The four fractions obtained were 0-4% PEG precipitate, 4-10% PEG precipitate, 10-20% PEG precipitate and 20% PEG supernatant. The 20% PEG supernatant fraction was dominated by albumin but also contained most of the API. However, this fraction also contained numerous other proteins, including all of the alpha-1-acid glycoprotein, antithrombin III, ceruloplasmin, haptoglobin, transferrin, Cl esterase inhibitor, prealbumin, retinol binding protein, transcortin, and angiotensinogen.
Several other groups have combined PEG precipitation with other purification methods in an attempt to isolate API. For instance, U.S. Pat. No. 4,379,087, U.S. Pat. No. 4,439,358, U.S. Pat. No. 4,697,003 and U.S. Pat. No. 4,656,254, all employ a PEG precipitation step in processes of isolating API. PEG precipitation is disadvantageous in that PEG will also precipitate hepatitus B virus. Although viruses are typically inactivated in a heat pasteurization step following purification of API, precaution must be taken, and patients must therefore be immunized before receiving treatments.
Along with PEG precipitation, U.S. Pat. No. 4,379,087 by Coan et al. also reports a concentration step involving phosphate buffer and DEAE SEPHAROSE.TM.. The combined process is quite lengthy, i.e., five days. Furthermore, the final product is only 60% active and only 80% pure.
Japanese Patent No. 8-99999 describes using PEG precipitation in combination with an SP-cation exchanger. The methods described therein do not separate fully active API from inactive API. The specific activity of fully active API should be 1.88 (using an Extinction coefficient 5.3), but the product achieved by this process only shows a relative activity of 1.0. Moreover, the best yield, achieved by combining PEG precipitation and SP-cation exchange steps, was only 50%, and does not appear to be easily scaled up to a commercial production level.
U.S. Pat. No. 3,293,236 describes a process for purifying API using citrate buffered cation exchange chromatography, and combines this step with fractionation of human plasma with ammonium sulfate. The importance of the DEAE purification step of the present invention becomes apparent when one examines the yields reported in U.S. Pat. No. 3,293,236. The yields were not measured by specific activity of API, and the reported quantities of protein equate to much more API than is initially present in human plasma. Thus, the product achieved by the method disclosed in U.S. Pat. No. 3,293,236 must have a high quantity of contaminating protein.
U.S. Pat. No. 5,610,285 discloses a purification process which combines successive anion and cation exchange chromatography steps. The initial anion exchange chromatography step binds API to the column; however, it also binds numerous contaminating proteins, particularly lipoproteins. Lipoproteins are plentiful in many of the materials from which API is isolated (e.g. Cohn IV paste), and so tend to occlude the column. Such occlusion requires columns of considerable size, additional dialysis/filtration steps, and at least two cation chromatography steps. Those requirements reduce efficiency and practicality of the method for large-scale processes.
Further, in the '285 process all API, both inactive and active protein, bind to the anion exchange column. And when the API is eluted from that column in accordance with that method, i.e., high salt phosphate buffer, both active and inactive protein come off the column. Thus, there is no separation of the active from the inactive protein.